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Research article
Rapid Identification of Staphylococcus Species Isolated from Food Samples by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
1Institute of Life Sciences and Resources and Department of Food Science and Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea
2Department of Bioinformatics and Biosystems, Korea Polytechnics, Seongnam 13122, Republic of Korea
J. Microbiol. Biotechnol. 2019; 29(4): 548-557
Published April 28, 2019 https://doi.org/10.4014/jmb.1901.01046
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
The
For the identification of
Recently, MALDI-TOF MS has emerged as a breakthrough means for the rapid and routine identification of micro- organisms with regard to cost-effectiveness, time-savings, high reproducibility, and high reliability [10-12]. MALDI- TOF MS technology depends on the generation of unique mass spectra captured from a small amount of microbial colony followed by comparison to a reference database containing known microbial spectra for identification of microorganisms [10,13-15]. Studies on clinical applications for pathogenic staphylococci diagnostics and on the identification of a variety of
In this present study, we evaluated the ability of MALDI- TOF MS to identify
Materials and Methods
Bacterial Strains
The
-
Table 1 .
Staphylococcus reference strains used in this study.Bacterial strains Strain designations or originsa Cluster groupb Staphylococcus aureus ATCC 6538, ATCC 6538P, ATCC 29737, NCCP 14560 S. aureus Staphylococcus epidermidis ATCC 12228, ATCC 14990, NCCP 14723 S. epidermidis Staphylococcus capitis NCCP 14663 S. epidermidis Staphylococcus caprae KCTC 3583 S. epidermidis Staphylococcus haemolyticus ATCC 29970 S. haemolyticus Staphylococcus hominis NCCP 10748 S. haemolyticus Staphylococcus schleiferi subsp.coagulans KCCM 41634 S. hyicus-intermedius Staphylococcus lugdunensis NCCP 15630 S. lugdunensis Staphylococcus saprophyticus NCCP 14670 S. saprophyticus Staphylococcus xylosus NCCP 10937 S. saprophyticus Staphylococcus lentus KCCM 41469 S. sciuri Staphylococcus sciuri subsp.sciuri KCCM 41468 S. sciuri Staphylococcus warneri KCTC 3340 S. warneri Staphylococcus pettenkoferi DSM 19554 - a ATCC, American Type Culture Collection; NCCP, National Culture Collection for Pathogens of Korea; KCTC, Korean Collection for Type Culture; KCCM, Korean Culture Center of Microorganisms; DSM, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures.
b Cluster Groups of
Staphylococcus species were described from the phylogenetic study based on 16S rRNA gene sequence analysis (Takahashiet al . 1999).
Isolation of Staphylococcus Species
Presumptive
Sample Preparation for MALDI-TOF MS
A loopful of each
For the direct transfer method (DT); a single colony was deposited directly on an assigned position of an MSP 96 target plate, followed by drying at ambient temperature. The dried sample was overlaid with 1 μl of HCCA matrix solution, followed by air-drying at ambient temperature for crystallizing.
For the extended direct transfer method (EDT); a single colony was deposited directly on an assigned position of an MSP 96 target plate and was immediately overlaid with 1 μl of 70% formic acid, then dried at ambient temperature. The sample was overlaid with 1 μl of HCCA matrix solution, followed by air-drying at ambient temperature for crystallizing.
For the full extraction method (EX); a loopful of a colony of each bacterium scraped from the agar plate was suspended in 300 μl of sterile distilled water in a micro-centrifuge tube, followed by the addition of 900 μl ethanol and vortexing. The bacterial suspension was centrifuged at 16,000 ×
MALDI-TOF MS Analysis
Bacterial identification of
16S rRNA Gene Sequencing
The
Results
MALDI-TOF MS Analysis of Staphylococcus Reference Strains
The diagnostic ability of MALDI-TOF MS was evaluated with 19 reference strains by incubation period (Day-1, Day-2, and Day-3) and by sample preparation method (DT, EDT, and EX) as shown in Tables 2 and 3. Overall diagnostic accuracy considering identification at the genus and species levels revealed that almost 19 reference strains were identified correctly. At the genus identification level, the EDT and EX sample preparation methods provided excellent identification results with almost 100% accuracy regardless of incubation period. Meanwhile, the DT method showed relatively low identification accuracy (Table 2). At the species identification level, the EX method at Day-1 revealed the best diagnostic ability (89% accuracy) (Table 3). The EDT method revealed a constant identification yield (between 78% to 89% accuracy) regardless of incubation period, whereas the DT method yielded low accuracy (less than 64% accuracy) with discrepant results. The relatively low accuracy seen when using the DT method may be due to the absence of formic acid treatment and the simple steps in the DT method that provide relatively low protein extraction from bacterial cell walls. Based on our comparison of these three preparation methods, the EX extraction method was selected for identification of
-
Table 2 . Performance of MALDI-TOF MS for the identification of
Staphylococcus reference strains at genus level (log scores ≥1.7) by sample preparation method and incubation period of bacteria culture.Strain Day 1 Day 2 Day 3 DTa EDT EX DT EDT EX DT EDT EX S. aureus (n=4)4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) S. epidermidis (n=3)3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) S. capitis (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. caprae (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. haemolyticus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. hominis (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. schleiferi (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. lugdunensis (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. saprophyticus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. xylosus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. lentus (n=1)1 (100%) 1 (100%) 1 (100%) 0 (0%) 0 (0%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. sciuri (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. warneri (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. pettenkoferi (n=1)0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) Total (n=19) 18 (94.7%) 19 (100%) 19 (100%) 18 (94.7%) 18 (94.7%) 19 (100%) 17 (89.5%) 19 (100%) 19 (100%) a DT, direct transfer method; EDT, extended direct transfer method; EX, extraction method.
-
Table 3 . Performance of MALDI-TOF MS for the identification of
Staphylococcus reference strains at species level (log scores ≥2.0) by sample preparation method and incubation period of bacteria culture.Strain Day 1 Day 2 Day 3 DT EDT EX DT EDT EX DT EDT EX S. aureus (n=4)4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) S. epidermidis (n=3)2 (66.7%) 3 (100%) 3 (100%) 1 (33.3%) 2 (66.7%) 2 (66.7%) 0 (0%) 3 (100%) 1 (33.3%) S. capitis (n=1)0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. caprae (n=1)0 (0%) 0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. haemolyticus (n=1)0 (0%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. hominis (n=1)0 (0%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. schleiferi (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. lugdunensis (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. saprophyticus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. xylosus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. lentus (n=1)0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) S. sciuri (n=1)1 (100%) 0 (0%) 0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) S. warneri (n=1)1 (100%) 0 (0%) 1 (100%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (100%) S. pettenkoferi (n=1)0 (0%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) 0 (0%) Total (n=19) 12 (63.2%) 15 (78.9%) 17 (89.5%) 12 (63.2%) 15 (78.9%) 15 (78.9%) 10 (52.6%) 17 (89.5%) 14 (73.7%)
The analyzed spectra obtained by MALDI-TOF MS and the MSP-based dendrogram for the 19 reference strains yielded by the EX method at Day-1, are shown in Figs. 1 and 2. The MALDI-TOF MS analysis yielded clearly different spectra. Their overall dendrogram (see Fig. 2) reveals clean distinctions between
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Fig. 1. MALDI-TOF MS spectra of 19 Staphylococcus reference strains in the 2,000 to 20,000 Da range.
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Fig. 2.
Main spectra profile (MSP)-based dendrogram from MALDI-TOF MS spectra of the 19 Distance values below the dendrogram are relative and normalized to a maximal value of 1,000.Staphylococcus reference strains and species clustering of the isolates analyzed with the reference strains.
MALDI-TOF MS Analysis of Staphylococcus Strains Isolated from Food Samples
A total of 96
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Table 4 . Identified population of
Staphylococcus species using MALDI-TOF MS analysis isolated from various foods in Korea.Food source Number of Staphylococcus speciesTotal S. capitis S. epidermidis S. equorum S. hominis S. Pasteuri S. simulans S. warneri Shrimp-jeotgala 3 12 16 0 8 12 1 52 Shellfish-jeotgal 3 6 0 0 1 0 0 10 Fish-jeotgal 0 10 0 1 0 0 0 11 Salted Chinese cabbageb 0 0 0 9 0 0 0 9 Raw milk 0 14 0 0 0 0 0 14 Total 6 42 16 10 9 12 1 96 a Jeotgal is traditional Korean fermented sea food (Shrimp-jeotgal, Shellfish-jeotgal, Fish-jeotgal).
b Salted Chinese cabbage is a major ingredient of kimchi, a traditional Korean fermented food.
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Table 5 . Identification of 96
Staphylococcus isolates by MALDI-TOF MS.Species (no. of isolates) No. of Staphylococcus species with resultsa:≥ 2.000 1.700-1.999 ≤ 1.699 S. epidermidis (42)40 2 0 S. pasteuri (9)9 0 0 S. capitis (6)6 0 0 S. hominis (10)7 3 0 S. equorum (16)4 12 0 S. simulans (12)11 1 0 S. warneri (1)1 0 0 Total isolates (96) 78 18 0 a ≥ 2.000: species-level-identification; 1.700~1.999: genus-level-identification; ≤ 1.699: not reliable identification.
PCA and MSP-Based Dendrogram of Isolated Staphylococcus
PCA using Biotyper software was accomplished to identify intra-species similarity and variation between the 96 isolated
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Fig. 3.
Two dimensional plots of the spectra of 96 The isolates are clearly separated into 6 groups, which visually demonstrates the heterogeneity of the protein spectra amongStaphylococcus isolates generated by principal component analysis (PCA).Staphylococcus species. Each dot indicates the spectrum of one isolate.
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Fig. 4.
Main spectra profile (MSP)-based dendrogram from MALDI-TOF MS spectra of the 96 Clusters are based on theStaphylococcus strains isolated from various foods.Staphylococcus species level. Distance values below the dendrogram are relative and normalized to a maximal value of 1,000.
16S rRNA Gene Sequencing
The identification results of comparing 16S rRNA gene sequencing and MALDI-TOF MS are listed in Table 6. Based on the 16S rRNA gene sequencing, 6 isolates were identified as
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Table 6 . Comparison of the identification results of
Staphylococcus isolates by 16S rRNA gene sequencing and MALDI-TOF MS.Origin MALDI-TOF MS results (na) 16S rRNA gene sequencing results (NCBI accession no.) Identity Shrimp-Jeotgal S. capitis (3)S. capitis (MK318575.1)100% S. epidermidis (12)S. epidermidis (KY753228.1)100% S. equorum (16)S. equorum (MK253324.1),S. haemolyticus (MF578766.1)100% S. pasteuri (8)S. pasteuri (KT036409.1),S. warneri (KT720133.1)100% S. simulans (12)S. simulans (KC849422.1)100% S. warneri (1)S. warneri (KT153529.1),S. pasteuri (KT427912.1)100% Shellfish-Jeotgal S. capitis (3)S. capitis (KT027728.1)100% S. epidermidis (6)S. epidermidis (MG645276.1)100% S. pasteuri (1)S. pasteuri (MH158278.1),S. warneri (MG920271.1)100% Fish-Jeotgal S. epidermidis (10)S. epidermidis (KT427443.1)100% S. hominis (1)S. hominis (MH715220.1)100% Salted Chinese cabbage S. hominis (9)S. hominis (MK318620.1)100% Raw milk S. epidermidis (14)S. epidermidis (MH118521.1)100% aNumber of isolates.
Discussion
A phylogenetic study of 38 species in the
In order to accurately identify
We evaluated the performance of MALDI-TOF MS for the identification of
We compared identification results of
The identified
In this study, the identification of isolated
Acknowledgments
This work was funded by the Strategic Initiative for Microbiomes in Agriculture and Food, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea (Grant number 918005-4).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2019; 29(4): 548-557
Published online April 28, 2019 https://doi.org/10.4014/jmb.1901.01046
Copyright © The Korean Society for Microbiology and Biotechnology.
Rapid Identification of Staphylococcus Species Isolated from Food Samples by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Eiseul Kim 1, Hyun-Joong Kim 1, Seung-Min Yang 1, Chang-Gyeom Kim 2, Dong-Won Choo 2 and Hae-Yeong Kim 1*
1Institute of Life Sciences and Resources and Department of Food Science and Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea
2Department of Bioinformatics and Biosystems, Korea Polytechnics, Seongnam 13122, Republic of Korea
Correspondence to:Hae-Yeong Kim
hykim@khu.ac.kr
Abstract
Staphylococcus species have a ubiquitous habitat in a wide range of foods, thus the ability to identify staphylococci at the species level is critical in the food industry. In this study, we performed rapid identification of Staphylococcus species using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS was evaluated for the identification of Staphylococcus reference strains (n = 19) and isolates (n = 96) from various foods with consideration for the impact of sample preparation methods and incubation period. Additionally, the spectra of isolated Staphylococcus strains were analyzed using principal component analysis (PCA) and a main spectra profile (MSP)-based dendrogram. MALDI-TOF MS accurately identified Staphylococcus reference strains and isolated strains: the highest performance was by the EX method (83.3~89.5% accuracy) at species level identification (EDT, 70.3~78.9% accuracy; DT, less than 46.3~63.2% accuracy) of 24-h cultured colonies. Identification results at the genus level were 100% accurate at EDT, EX sample preparation and 24-h incubation time. On the other hand, the DT method showed relatively low identification accuracy in all extraction methods and incubation times. The analyzed spectra and MSP-based dendrogram showed that the isolated Staphylococcus strains were characterized at the species level. The performance analysis of MALDI-TOF MS shows the method has the potential ability to discriminate between Staphylococcus species from foods in Korea. This study provides valuable information that MALDI-TOF MS can be applied to monitor microbial populations and pathogenic bacteria in the food industry thereby contributing to food safety.
Keywords: MALDI-TOF MS, Staphylococcus, jeotgal, fermented food, identification, extraction method
Introduction
The
For the identification of
Recently, MALDI-TOF MS has emerged as a breakthrough means for the rapid and routine identification of micro- organisms with regard to cost-effectiveness, time-savings, high reproducibility, and high reliability [10-12]. MALDI- TOF MS technology depends on the generation of unique mass spectra captured from a small amount of microbial colony followed by comparison to a reference database containing known microbial spectra for identification of microorganisms [10,13-15]. Studies on clinical applications for pathogenic staphylococci diagnostics and on the identification of a variety of
In this present study, we evaluated the ability of MALDI- TOF MS to identify
Materials and Methods
Bacterial Strains
The
-
Table 1 .
Staphylococcus reference strains used in this study..Bacterial strains Strain designations or originsa Cluster groupb Staphylococcus aureus ATCC 6538, ATCC 6538P, ATCC 29737, NCCP 14560 S. aureus Staphylococcus epidermidis ATCC 12228, ATCC 14990, NCCP 14723 S. epidermidis Staphylococcus capitis NCCP 14663 S. epidermidis Staphylococcus caprae KCTC 3583 S. epidermidis Staphylococcus haemolyticus ATCC 29970 S. haemolyticus Staphylococcus hominis NCCP 10748 S. haemolyticus Staphylococcus schleiferi subsp.coagulans KCCM 41634 S. hyicus-intermedius Staphylococcus lugdunensis NCCP 15630 S. lugdunensis Staphylococcus saprophyticus NCCP 14670 S. saprophyticus Staphylococcus xylosus NCCP 10937 S. saprophyticus Staphylococcus lentus KCCM 41469 S. sciuri Staphylococcus sciuri subsp.sciuri KCCM 41468 S. sciuri Staphylococcus warneri KCTC 3340 S. warneri Staphylococcus pettenkoferi DSM 19554 - a ATCC, American Type Culture Collection; NCCP, National Culture Collection for Pathogens of Korea; KCTC, Korean Collection for Type Culture; KCCM, Korean Culture Center of Microorganisms; DSM, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. .
b Cluster Groups of
Staphylococcus species were described from the phylogenetic study based on 16S rRNA gene sequence analysis (Takahashiet al . 1999)..
Isolation of Staphylococcus Species
Presumptive
Sample Preparation for MALDI-TOF MS
A loopful of each
For the direct transfer method (DT); a single colony was deposited directly on an assigned position of an MSP 96 target plate, followed by drying at ambient temperature. The dried sample was overlaid with 1 μl of HCCA matrix solution, followed by air-drying at ambient temperature for crystallizing.
For the extended direct transfer method (EDT); a single colony was deposited directly on an assigned position of an MSP 96 target plate and was immediately overlaid with 1 μl of 70% formic acid, then dried at ambient temperature. The sample was overlaid with 1 μl of HCCA matrix solution, followed by air-drying at ambient temperature for crystallizing.
For the full extraction method (EX); a loopful of a colony of each bacterium scraped from the agar plate was suspended in 300 μl of sterile distilled water in a micro-centrifuge tube, followed by the addition of 900 μl ethanol and vortexing. The bacterial suspension was centrifuged at 16,000 ×
MALDI-TOF MS Analysis
Bacterial identification of
16S rRNA Gene Sequencing
The
Results
MALDI-TOF MS Analysis of Staphylococcus Reference Strains
The diagnostic ability of MALDI-TOF MS was evaluated with 19 reference strains by incubation period (Day-1, Day-2, and Day-3) and by sample preparation method (DT, EDT, and EX) as shown in Tables 2 and 3. Overall diagnostic accuracy considering identification at the genus and species levels revealed that almost 19 reference strains were identified correctly. At the genus identification level, the EDT and EX sample preparation methods provided excellent identification results with almost 100% accuracy regardless of incubation period. Meanwhile, the DT method showed relatively low identification accuracy (Table 2). At the species identification level, the EX method at Day-1 revealed the best diagnostic ability (89% accuracy) (Table 3). The EDT method revealed a constant identification yield (between 78% to 89% accuracy) regardless of incubation period, whereas the DT method yielded low accuracy (less than 64% accuracy) with discrepant results. The relatively low accuracy seen when using the DT method may be due to the absence of formic acid treatment and the simple steps in the DT method that provide relatively low protein extraction from bacterial cell walls. Based on our comparison of these three preparation methods, the EX extraction method was selected for identification of
-
Table 2 . Performance of MALDI-TOF MS for the identification of
Staphylococcus reference strains at genus level (log scores ≥1.7) by sample preparation method and incubation period of bacteria culture. .Strain Day 1 Day 2 Day 3 DTa EDT EX DT EDT EX DT EDT EX S. aureus (n=4)4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) S. epidermidis (n=3)3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) S. capitis (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. caprae (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. haemolyticus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. hominis (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. schleiferi (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. lugdunensis (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. saprophyticus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. xylosus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. lentus (n=1)1 (100%) 1 (100%) 1 (100%) 0 (0%) 0 (0%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. sciuri (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. warneri (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. pettenkoferi (n=1)0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) Total (n=19) 18 (94.7%) 19 (100%) 19 (100%) 18 (94.7%) 18 (94.7%) 19 (100%) 17 (89.5%) 19 (100%) 19 (100%) a DT, direct transfer method; EDT, extended direct transfer method; EX, extraction method..
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Table 3 . Performance of MALDI-TOF MS for the identification of
Staphylococcus reference strains at species level (log scores ≥2.0) by sample preparation method and incubation period of bacteria culture. .Strain Day 1 Day 2 Day 3 DT EDT EX DT EDT EX DT EDT EX S. aureus (n=4)4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) S. epidermidis (n=3)2 (66.7%) 3 (100%) 3 (100%) 1 (33.3%) 2 (66.7%) 2 (66.7%) 0 (0%) 3 (100%) 1 (33.3%) S. capitis (n=1)0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. caprae (n=1)0 (0%) 0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. haemolyticus (n=1)0 (0%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. hominis (n=1)0 (0%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. schleiferi (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. lugdunensis (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. saprophyticus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. xylosus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. lentus (n=1)0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) S. sciuri (n=1)1 (100%) 0 (0%) 0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) S. warneri (n=1)1 (100%) 0 (0%) 1 (100%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (100%) S. pettenkoferi (n=1)0 (0%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) 0 (0%) Total (n=19) 12 (63.2%) 15 (78.9%) 17 (89.5%) 12 (63.2%) 15 (78.9%) 15 (78.9%) 10 (52.6%) 17 (89.5%) 14 (73.7%)
The analyzed spectra obtained by MALDI-TOF MS and the MSP-based dendrogram for the 19 reference strains yielded by the EX method at Day-1, are shown in Figs. 1 and 2. The MALDI-TOF MS analysis yielded clearly different spectra. Their overall dendrogram (see Fig. 2) reveals clean distinctions between
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Figure 1. MALDI-TOF MS spectra of 19 Staphylococcus reference strains in the 2,000 to 20,000 Da range.
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Figure 2.
Main spectra profile (MSP)-based dendrogram from MALDI-TOF MS spectra of the 19 Distance values below the dendrogram are relative and normalized to a maximal value of 1,000.Staphylococcus reference strains and species clustering of the isolates analyzed with the reference strains.
MALDI-TOF MS Analysis of Staphylococcus Strains Isolated from Food Samples
A total of 96
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Table 4 . Identified population of
Staphylococcus species using MALDI-TOF MS analysis isolated from various foods in Korea..Food source Number of Staphylococcus speciesTotal S. capitis S. epidermidis S. equorum S. hominis S. Pasteuri S. simulans S. warneri Shrimp-jeotgala 3 12 16 0 8 12 1 52 Shellfish-jeotgal 3 6 0 0 1 0 0 10 Fish-jeotgal 0 10 0 1 0 0 0 11 Salted Chinese cabbageb 0 0 0 9 0 0 0 9 Raw milk 0 14 0 0 0 0 0 14 Total 6 42 16 10 9 12 1 96 a Jeotgal is traditional Korean fermented sea food (Shrimp-jeotgal, Shellfish-jeotgal, Fish-jeotgal)..
b Salted Chinese cabbage is a major ingredient of kimchi, a traditional Korean fermented food..
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Table 5 . Identification of 96
Staphylococcus isolates by MALDI-TOF MS..Species (no. of isolates) No. of Staphylococcus species with resultsa:≥ 2.000 1.700-1.999 ≤ 1.699 S. epidermidis (42)40 2 0 S. pasteuri (9)9 0 0 S. capitis (6)6 0 0 S. hominis (10)7 3 0 S. equorum (16)4 12 0 S. simulans (12)11 1 0 S. warneri (1)1 0 0 Total isolates (96) 78 18 0 a ≥ 2.000: species-level-identification; 1.700~1.999: genus-level-identification; ≤ 1.699: not reliable identification..
PCA and MSP-Based Dendrogram of Isolated Staphylococcus
PCA using Biotyper software was accomplished to identify intra-species similarity and variation between the 96 isolated
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Figure 3.
Two dimensional plots of the spectra of 96 The isolates are clearly separated into 6 groups, which visually demonstrates the heterogeneity of the protein spectra amongStaphylococcus isolates generated by principal component analysis (PCA).Staphylococcus species. Each dot indicates the spectrum of one isolate.
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Figure 4.
Main spectra profile (MSP)-based dendrogram from MALDI-TOF MS spectra of the 96 Clusters are based on theStaphylococcus strains isolated from various foods.Staphylococcus species level. Distance values below the dendrogram are relative and normalized to a maximal value of 1,000.
16S rRNA Gene Sequencing
The identification results of comparing 16S rRNA gene sequencing and MALDI-TOF MS are listed in Table 6. Based on the 16S rRNA gene sequencing, 6 isolates were identified as
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Table 6 . Comparison of the identification results of
Staphylococcus isolates by 16S rRNA gene sequencing and MALDI-TOF MS..Origin MALDI-TOF MS results (na) 16S rRNA gene sequencing results (NCBI accession no.) Identity Shrimp-Jeotgal S. capitis (3)S. capitis (MK318575.1)100% S. epidermidis (12)S. epidermidis (KY753228.1)100% S. equorum (16)S. equorum (MK253324.1),S. haemolyticus (MF578766.1)100% S. pasteuri (8)S. pasteuri (KT036409.1),S. warneri (KT720133.1)100% S. simulans (12)S. simulans (KC849422.1)100% S. warneri (1)S. warneri (KT153529.1),S. pasteuri (KT427912.1)100% Shellfish-Jeotgal S. capitis (3)S. capitis (KT027728.1)100% S. epidermidis (6)S. epidermidis (MG645276.1)100% S. pasteuri (1)S. pasteuri (MH158278.1),S. warneri (MG920271.1)100% Fish-Jeotgal S. epidermidis (10)S. epidermidis (KT427443.1)100% S. hominis (1)S. hominis (MH715220.1)100% Salted Chinese cabbage S. hominis (9)S. hominis (MK318620.1)100% Raw milk S. epidermidis (14)S. epidermidis (MH118521.1)100% aNumber of isolates..
Discussion
A phylogenetic study of 38 species in the
In order to accurately identify
We evaluated the performance of MALDI-TOF MS for the identification of
We compared identification results of
The identified
In this study, the identification of isolated
Acknowledgments
This work was funded by the Strategic Initiative for Microbiomes in Agriculture and Food, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea (Grant number 918005-4).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

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Table 1 .
Staphylococcus reference strains used in this study..Bacterial strains Strain designations or originsa Cluster groupb Staphylococcus aureus ATCC 6538, ATCC 6538P, ATCC 29737, NCCP 14560 S. aureus Staphylococcus epidermidis ATCC 12228, ATCC 14990, NCCP 14723 S. epidermidis Staphylococcus capitis NCCP 14663 S. epidermidis Staphylococcus caprae KCTC 3583 S. epidermidis Staphylococcus haemolyticus ATCC 29970 S. haemolyticus Staphylococcus hominis NCCP 10748 S. haemolyticus Staphylococcus schleiferi subsp.coagulans KCCM 41634 S. hyicus-intermedius Staphylococcus lugdunensis NCCP 15630 S. lugdunensis Staphylococcus saprophyticus NCCP 14670 S. saprophyticus Staphylococcus xylosus NCCP 10937 S. saprophyticus Staphylococcus lentus KCCM 41469 S. sciuri Staphylococcus sciuri subsp.sciuri KCCM 41468 S. sciuri Staphylococcus warneri KCTC 3340 S. warneri Staphylococcus pettenkoferi DSM 19554 - a ATCC, American Type Culture Collection; NCCP, National Culture Collection for Pathogens of Korea; KCTC, Korean Collection for Type Culture; KCCM, Korean Culture Center of Microorganisms; DSM, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. .
b Cluster Groups of
Staphylococcus species were described from the phylogenetic study based on 16S rRNA gene sequence analysis (Takahashiet al . 1999)..
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Table 2 . Performance of MALDI-TOF MS for the identification of
Staphylococcus reference strains at genus level (log scores ≥1.7) by sample preparation method and incubation period of bacteria culture. .Strain Day 1 Day 2 Day 3 DTa EDT EX DT EDT EX DT EDT EX S. aureus (n=4)4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) S. epidermidis (n=3)3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) 3 (100%) S. capitis (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. caprae (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. haemolyticus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. hominis (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. schleiferi (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. lugdunensis (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. saprophyticus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. xylosus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. lentus (n=1)1 (100%) 1 (100%) 1 (100%) 0 (0%) 0 (0%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. sciuri (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. warneri (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. pettenkoferi (n=1)0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) Total (n=19) 18 (94.7%) 19 (100%) 19 (100%) 18 (94.7%) 18 (94.7%) 19 (100%) 17 (89.5%) 19 (100%) 19 (100%) a DT, direct transfer method; EDT, extended direct transfer method; EX, extraction method..
-
Table 3 . Performance of MALDI-TOF MS for the identification of
Staphylococcus reference strains at species level (log scores ≥2.0) by sample preparation method and incubation period of bacteria culture. .Strain Day 1 Day 2 Day 3 DT EDT EX DT EDT EX DT EDT EX S. aureus (n=4)4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) 4 (100%) S. epidermidis (n=3)2 (66.7%) 3 (100%) 3 (100%) 1 (33.3%) 2 (66.7%) 2 (66.7%) 0 (0%) 3 (100%) 1 (33.3%) S. capitis (n=1)0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. caprae (n=1)0 (0%) 0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. haemolyticus (n=1)0 (0%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. hominis (n=1)0 (0%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. schleiferi (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. lugdunensis (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) S. saprophyticus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. xylosus (n=1)1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) S. lentus (n=1)0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) S. sciuri (n=1)1 (100%) 0 (0%) 0 (0%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 1 (100%) 0 (0%) S. warneri (n=1)1 (100%) 0 (0%) 1 (100%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (100%) S. pettenkoferi (n=1)0 (0%) 1 (100%) 1 (100%) 0 (0%) 1 (100%) 0 (0%) 1 (100%) 1 (100%) 0 (0%) Total (n=19) 12 (63.2%) 15 (78.9%) 17 (89.5%) 12 (63.2%) 15 (78.9%) 15 (78.9%) 10 (52.6%) 17 (89.5%) 14 (73.7%)
-
Table 4 . Identified population of
Staphylococcus species using MALDI-TOF MS analysis isolated from various foods in Korea..Food source Number of Staphylococcus speciesTotal S. capitis S. epidermidis S. equorum S. hominis S. Pasteuri S. simulans S. warneri Shrimp-jeotgala 3 12 16 0 8 12 1 52 Shellfish-jeotgal 3 6 0 0 1 0 0 10 Fish-jeotgal 0 10 0 1 0 0 0 11 Salted Chinese cabbageb 0 0 0 9 0 0 0 9 Raw milk 0 14 0 0 0 0 0 14 Total 6 42 16 10 9 12 1 96 a Jeotgal is traditional Korean fermented sea food (Shrimp-jeotgal, Shellfish-jeotgal, Fish-jeotgal)..
b Salted Chinese cabbage is a major ingredient of kimchi, a traditional Korean fermented food..
-
Table 5 . Identification of 96
Staphylococcus isolates by MALDI-TOF MS..Species (no. of isolates) No. of Staphylococcus species with resultsa:≥ 2.000 1.700-1.999 ≤ 1.699 S. epidermidis (42)40 2 0 S. pasteuri (9)9 0 0 S. capitis (6)6 0 0 S. hominis (10)7 3 0 S. equorum (16)4 12 0 S. simulans (12)11 1 0 S. warneri (1)1 0 0 Total isolates (96) 78 18 0 a ≥ 2.000: species-level-identification; 1.700~1.999: genus-level-identification; ≤ 1.699: not reliable identification..
-
Table 6 . Comparison of the identification results of
Staphylococcus isolates by 16S rRNA gene sequencing and MALDI-TOF MS..Origin MALDI-TOF MS results (na) 16S rRNA gene sequencing results (NCBI accession no.) Identity Shrimp-Jeotgal S. capitis (3)S. capitis (MK318575.1)100% S. epidermidis (12)S. epidermidis (KY753228.1)100% S. equorum (16)S. equorum (MK253324.1),S. haemolyticus (MF578766.1)100% S. pasteuri (8)S. pasteuri (KT036409.1),S. warneri (KT720133.1)100% S. simulans (12)S. simulans (KC849422.1)100% S. warneri (1)S. warneri (KT153529.1),S. pasteuri (KT427912.1)100% Shellfish-Jeotgal S. capitis (3)S. capitis (KT027728.1)100% S. epidermidis (6)S. epidermidis (MG645276.1)100% S. pasteuri (1)S. pasteuri (MH158278.1),S. warneri (MG920271.1)100% Fish-Jeotgal S. epidermidis (10)S. epidermidis (KT427443.1)100% S. hominis (1)S. hominis (MH715220.1)100% Salted Chinese cabbage S. hominis (9)S. hominis (MK318620.1)100% Raw milk S. epidermidis (14)S. epidermidis (MH118521.1)100% aNumber of isolates..
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